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Molecular Regulation of Cardiomyocyte Differentiation

Sharon L. Paige1, Karolina Plonowska2, Adele Xu2, and Sean M. Wu1,2,3,4,5

1Department of Pediatrics and Division of Pediatric Cardiology, Stanford University School of Medicine, Stanford, CA, 94305, USA

2Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, 94305, USA

3Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA

4Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, 94305, USA

5Child Health Research Institute, Stanford University School of Medicine, Stanford, CA, 94305, USA

Abstract

The heart is the first organ to form during embryonic development. Given the complex nature of

cardiac differentiation and morphogenesis, it is not surprising that some form of congenital heart

disease is present in approximately one percent of newborns. The molecular determinants of heart

development have received much attention over the past several decades. This has been driven in

large part by an interest in understanding the etiology of congenital heart disease coupled with the

potential of utilizing knowledge from developmental biology to generate functional cells and

tissues that could be used for regenerative medicine purposes. In this review, we highlight the

critical signaling pathways and transcription factor networks that regulate cardiomyocyte lineage

specification in both in vivo and in vitro models. Special focus will be given to epigenetic

regulators that drive the commitment of cardiomyogenic cells from nascent mesoderm and their

differentiation into chamber-specific myocytes as well as regulation of myocardial trabeculation.

Keywords

cardiac development; cardiomyocyte; transcription factor; molecular regulation

Introduction

Molecular regulation of heart formation has fascinated biologists for more than a century.

Driven by both the need for greater understanding of the etiology of congenital heart disease

and its potential in regenerative medicine, investigators in cardiac developmental biology

Correspondence: Sean M. Wu, MD, PhD, Lokey Stem Cell Research Building, Rm G1120A, 265 Campus Dr. Stanford, CA 94305, P: 650-724-4498, F: 650-724-4689, [email protected].

Financial Disclosure and Conflict of InterestNone

HHS Public AccessAuthor manuscriptCirc Res. Author manuscript; available in PMC 2016 January 16.

Published in final edited form as:Circ Res. 2015 January 16; 116(2): 341–353. doi:10.1161/CIRCRESAHA.116.302752.

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have devoted significant efforts in recent years to address fundamental mechanisms that

underlie the complex patterning of the developing heart. In this review, we aim to

summarize succinctly the critical regulators of this process. In particular, we focus on

interactions between signaling molecules and transcription factor networks during

cardiomyocyte lineage commitment and myocardial chamber specification. Furthermore, we

highlight recent articles in this area and draw particular attention to epigenetic factors that

play a role in these processes. For a detailed discussion of earlier works on this topic, we

recommend several excellent reviews that address myocardial development in different

model organisms including mouse, fish, frogs, and chick.1, 2 We hope this article will inform

and inspire greater interest in cardiac development among investigators in cardiovascular

biology and spur new collaborations between basic and clinical scientists interested in

congenital heart disease.

Overview of Morphological Development

The following overview is intended to orient the reader with respect to major morphological

processes that will be discussed in further detail in subsequent sections. The heart is formed

from bilateral heart fields that are established during the gastrulation process (see Figure 1).

The heart field precursors migrate anteriorly and laterally from the primitive streak to form

the antero-lateral plate mesoderm which undergoes rapid migration towards the midline.

Subsequent fusion at the midline, led by closure of the foregut,3 gives rise to the linear heart

tube. Notably, elongation of the heart tube occurs primarily via addition of second heart

field (SHF)-derived cardiomyocytes to both poles, producing an embryonic organ that

consists of the truncus arteriosus most cranially followed by the bulbus cordis, ventricle,

atrium and sinus venosus. It is at this stage that the heart becomes functional as blood is

pumped from the venous pole to the arterial pole.4 The heart’s ability to generate blood flow

at this stage is critical to the continuation of cardiogenesis and to other processes in

embryogenesis that respond to hemodynamic signals.5–9 The process of rightward looping

occurs in conjunction with the addition of the SHF cells to both poles of the linear heart

tube. Concurrently, the developing atria migrate and become cranially positioned relative to

the ventricles. Complex remodeling, selective proliferation, myocardial subpopulation

specification, septation, and valve development then result in the formation of the four-

chambered heart.

Specification of cardiac mesoderm and formation of the cardiac crescent

During gastrulation, the three primary germ layers are formed with mesoderm sandwiched

between ventral endoderm and dorsal ectoderm. Myocardial precursor cells expressing the

T-box transcription factor T (Brachyury) migrate from the posterior epiblast through the

anterior portion of the primitive streak. These cells subsequently move laterally and, as the

embryonic coelom forms, will occupy bilateral regions of lateral splanchnic mesoderm that

is adjacent to underlying endoderm.10 The population of cardiac progenitor cells then

converges at the midline and organizes into a crescent, with the peak of the crescent located

cranially and the tails of the crescent extending caudally. Pro-cardiogenic signals from

underlying foregut endoderm and anti-cardiogenic signals from midline structures and the

neural plate determine the precise size and shape of the heart fields (Figure 2).10

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A cascade of transcription factor activation ultimately results in cardiac specification and

differentiation. During the primitive streak stage of mouse embryonic development, the T-

box transcription factor Eomesodermin (Eomes) activates the basic helix-loop-helix

transcription factor Mesoderm Posterior 1 (Mesp1).11, 12 Mesp1 expression at the start of

gastrulation represents the first known molecular step towards cardiogenesis. Mesp1 and

family member Mesp2 are the earliest markers of cardiovascular specification in the

developing embryo.13 The mechanism by which Mesp1 promotes cardiac specification is

multi-faceted in that Mesp1 drives expression of cardiac transcription factors while also

repressing genes that maintain pluripotency.14 As cardiac precursors migrate away from the

primitive streak to form the cardiac crescent, they down-regulate Mesp1 and Mesp2 while

activating other transcription factor networks that drive cardiac specification.

Among the pro-cardiogenic factors expressed in endoderm and mesoderm are Hedgehog,

bone morphogenetic proteins (BMPs)15, 16, fibroblast growth factors (FGFs), and non-

canonical Wnt/JNK.17 Canonical Wnt ligands, including Wnt1 and Wnt3a, secreted from the

neural tube inhibit cardiac mesoderm specification18, as do BMP antagonists Noggin and

Chordin secreted from the notochord. Canonical Wnt/β-catenin inhibitors, including Dkk-1

and crescent, are also secreted by the endoderm underlying cardiac mesoderm and serve to

counteract the inhibitory Wnt signals emanating from the neural plate.19, 20 Interestingly,

selective elimination of canonical Wnt/β-catenin signaling in endoderm via deletion of β-

catenin results in formation of ectopic cardiac tissue in overlying mesoderm.21 The

mechanism by which inhibition of Wnt/β-catenin signaling in endoderm induces cardiac

specification is thought to be due to activation of the homeodomain transcription factor Hex

in endoderm.22 Notably, Hex is associated with induction of cardiac transcription factors

such as Nkx2-5 and Tbx5 but does not directly activate expression of cardiac contractile

genes, such as those encoding myofilament proteins.22

First and Second Heart Fields

The earliest cells to express Mesp1 form the first heart field (FHF), which makes up the

cardiac crescent. A later wave of Mesp1 produces cardiac progenitors of the second heart

field (SHF), which are derived from pharyngeal mesoderm and lie anterior and medial to the

cells of the FHF.23–26 Clonal analysis in mice suggests that these progenitor cells are

committed to the FHF or SHF early, likely prior to the onset of Mesp1 expression.27 As the

cells of the FHF differentiate and proliferate during the formation of the linear heart tube,

the cells of the SHF retain their undifferentiated myocardial progenitor identity and become

positioned dorsally relative to the heart tube28. Lineage tracing studies in both chick and

mouse embryos have demonstrated that the cells of the FHF contribute primarily to the left

ventricle with small contributions to the atria while the SHF will form the right ventricle,

outflow tract, atria and inflow myocardium (see Figure 3).24, 25, 29–32 Within the SHF,

progenitor cells that will give rise to the right ventricle and outflow tract are considered

anterior SHF, while precursors of the atria and inflow tract are termed posterior SHF.33 SHF

progenitors demonstrate increased proliferation and delayed differentiation compared to

their FHF counterparts34 and differentiate as they contribute to both the venous and arterial

poles of the linear heart tube, promoting its elongation.

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The FHF cells are identified by the expression of Tbx5 and the first wave of Nkx2-5.35, 36

Recently, early expression of ion channel gene HCN4 was also reported to be FHF-specific,

leading to identification of the atrioventricular (AV) node and parts of the early conduction

system as FHF-derived structures.37, 38 Of note, early FHF-specific HCN4 expression is

distinct from later expression in the cardiac conduction system and sinoatrial node.37 The

second heart field is marked by expression of the LIM homeodomain transcription factor

Islet1 (Isl1).31, 35, 39 Recently, expression of Tbx1 in the second heart field was shown to

coordinate SHF contribution to the two poles of the linear heart tube.40 Other genes

expressed in SHF progenitor cells include Prdm1, Pitx2, Six1, Fgf8, and

Fgf10.28, 31–33, 41, 42 As cardiac differentiation progresses during addition of SHF cells to

the linear heart tube, these genes will be down-regulated as the next wave of cardiac

transcription factors becomes activated, including Nkx2-5, Gata4 and Mef2c.24, 32 A

summary of the key transcription factor interactions that regulate FHF and SHF

differentiation is shown in Figure 4.

In both human and mouse embryonic stem (ES) cell models, Isl1-expressing cells have been

shown to give rise to cardiomyocytes as well as smooth muscle cells and endothelium

(Figure 3).39, 43 The existence of a multipotent Isl1+ progenitor cell in vivo is postulated

from the contribution of Isl1-expressing SHF to the smooth muscle cells of the great vessels

(aorta and pulmonary trunk) and endothelial cells of the proximal coronary artery. However,

limited data exist to support the diversification of cardiovascular lineage cells from a single

Isl1+ cell. Nevertheless, a population of Isl1+ progenitors has been identified in both mouse

and human hearts in the fetal and early postnatal stages.43, 44 Reportedly, these cells

represent residual SHF progenitor cells given the significant decline in the number of these

cells after early fetal development and the absence of these cells in the adult heart. The

potential that these cells may participate in myocardial injury repair has motivated a number

of investigators to examine the presence of Isl1+ cells in the heart after infarction.45 Thus

far, no reactivation of Isl1+ cells or their contribution to new cardiomyocyte formation has

been found.

The characteristic delayed differentiation and continued proliferation of SHF progenitors are

regulated by FGF, Hedgehog and canonical Wnt/β- catenin signaling pathways. Fgf8 has

been identified as a necessary signaling molecule for SHF deployment as evidenced by the

observation that conditional ablation of Fgf8 in mesoderm results in formation of hearts that

are without a right ventricle or outflow tract.46 Other FGF family members involved in SHF

development include Fgf1047 and Fgf3.48 Shh is expressed in adjacent pharyngeal endoderm

and is required for SHF proliferation.49 Interestingly, Shh has been shown to activate a set

of forkhead-containing transcription factors including Foxa2, Foxc1 and Foxc2, which then

activate Tbx1.50, 51 Tbx1 in turn is critical for activation of Fgf3, Fgf8 and Fgf10, and for

suppression of Mef2c and SRF.46, 47, 52–54 Canonical Wnt/β- catenin pathways activate

proliferation in SHF cells in mouse embryos and also promote maintenance of multipotency

of Isl1+ progenitors derived from ES cells.43, 55

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Neural Crest and Proepicardial Contributions

Other cell lineages also contribute significantly to heart development alongside the first and

second heart fields. Classically, the postotic neural crest has been described to give rise to a

population of cells, known as the cardiac neural crest, that migrate from the neural tube in a

dorsolateral fashion to reach the caudal pharyngeal arches, from where they continue on to

migrate into the cardiac outflow tract.56 These cells are responsible for establishing the

aorticopulmonary septum, which divides the outflow tract into the aorta and pulmonary

artery, and give rise to the smooth muscle cells in the tunica media of both vessels in the

distal region of the arterial pole.29, 57 Recently, Arima et al. have demonstrated that the

preotic neural crest also contributes to the conotruncal region and interventricular septum.58

In addition, Escot et al. have identified SDF1 as a chemotactic agent driving the cardiac

neural crest cell migration towards the pharyngeal arches.59 Further, Holler et al. observed a

marked reduction in the neural crest-derived cells found in the outflow tract following loss

of Hand2 expression in murine embryos.60 Ablation of these cells results in persistent

truncus arteriosus, arterial pole alignment defects, and aberrant myocardium calcium

transients stemming from elevated Fgf8 expression, which normally appears to be

modulated by the cardiac neural crest cells.61 In addition, cardiac neural crest will contribute

the autonomic and sensory innervation of the heart. Anomalies involving the cardiac neural

crest cells are of clinical interest as they are responsible for a multitude of human

cardiocraniofacial defects, such as DiGeorge Syndrome (DGS) and velocardiofacial

syndrome (VCFS) due to the loss of Tbx1 in the context of 22q11 deletion.62 The

deployment of cardiac neural crest cells in a coordinated fashion and their dependence on

exchanges of signal with SHF cells is highlighted by the requirement of Jagged-1/Notch

signaling in the SHF cells to regulate Fgf8 expression that is essential for cardiac neural

crest cell migration and endothelial-mesenchymal transition. The exogenous replacement of

Fgf8 appears to rescue the developmental defect in explant assay of endocardial

cushion.63, 64

Cells of the proepicardial organ migrate to cover the surface of the developing heart. Though

little is known about the molecular drivers of this migration, studies in mouse embryos

suggest that the interplay between the α4 integrins and vascular adhesion molecule 1

(VCAM-1) is essential in maintaining the epicardial lining after embryonic day 11.65, 66

Some of these cells undergo an epithelial-to-mesenchymal transition and invade the

underlying myocardium. Del Monte et al. have shown that Notch1 activity may play a

critical role in this process by promoting blood vessel progenitor migration through the

compact myocardium.67 These cells then differentiate into cardiac fibroblasts and vascular

smooth muscle cells of the coronary vessels.68, 69 Specific subpopulations from within the

proepicardium may also contribute to coronary endothelium.70 Interestingly, two studies

report the differentiation of epicardial cells into cardiomyocytes,71, 72 however, significant

controversies exist in this area. For additional discussion, the readers are encouraged to

review articles that cover these lineages in greater detail.73–76

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Specification of Chamber Myocardium

The cardiomyocytes that make up the linear heart tube are termed “primary myocardium”

based on their slow proliferation rate, limited automaticity, slow conduction velocity and

poor contractility.77, 78 The cardiomyocytes that make up the outflow tract, inner curvature,

atrioventricular canal and sinus horns retain this primary phenotype. As atrial and

ventricular cells selectively proliferate and differentiate, “ballooning” of the outer curvature

of the heart tube occurs. The linear heart tube’s peristaltic pumping may drive this

ballooning process, as endocardial and myocardial cells alter their shape, size, and

proliferation in response to mechanical stress from blood flow and contraction.79–81 The

atrial and ventricular cells obtain a chamber myocardium identity, with associated fast

conduction velocity, increased automaticity, increased contractility and enhanced sarcomere

organization.82

Multiple transcription factors have been shown to regulate cardiac chamber morphogenesis

including the T-box transcription factors Tbx2, Tbx3, Tbx5 and Tbx20 as well as Nkx2-5,

Gata4, Cited1, Irx4, Irx1/Irx3/Irx5 and Hand1 (Figure 5). Tbx5 and Tbx20 act in

combination with Nkx2-5 and Gata4 to promote specification of chamber myocardium,

which includes induction of atrial natriuretic factor (encoded by Nppa), gap junction

proteins connexin 40 (Cx40) and connexin 43 (Cx43) as well as the cytoskeletal protein

Chisel (encoded by Smpx).4, 82, 83 Additionally, Tbx5 cooperates with Nkx2-5 to regulate

expression of the transcriptional repressor Id2, which contributes to patterning of the right

and left ventricular bundle branches.84, 85 The multiple roles of Tbx5 are consistent with the

varied cardiac phenotypes observed in patients with Holt-Oram syndrome.86 In contrast,

Tbx2 and Tbx3 can also interact with Nkx2-5 and Gata4 to repress chamber

specification.87, 88 Suppression of chamber specification by Tbx3 allows for the expression

of the pacemaker channel HCN4 in cells at the sinoatrial junction that will give rise to the

sinoatrial node.85, 89

Regions of chamber versus primary myocardium are exquisitely regulated by overlapping

regions of these various T-box transcription factors, leading to the proposed T-box code

hypothesis.1 Whereas Nkx2-5 and Gata4 are highly expressed throughout the linear heart

tube, the T-box transcription factors have more narrow expression patterns.78, 83 As a result

of retinoic acid signaling, Tbx5 is expressed in a graded fashion with peak expression

caudally in the area of the inflow tract and primitive atrium with diminished expression in

the primitive ventricle and absence in the outflow tract.86, 90 Tbx2 and Tbx3 are expressed in

the primary myocardium of the outflow tract, inner curvature, AV canal and inflow tract,

with Tbx2 expression extending more rostrally compared to Tbx3.78 Tbx2 has been shown to

inhibit proliferation and suppress expression of chamber myocardium-specific genes

including Cx40, Cx43, Smpx and Nppa.87, 91 Notably, Tbx20 is broadly expressed

throughout the linear heart tube and deletion of Tbx20 in mice results in defects in chamber

development, at least in part due to expansion of Tbx2 expression (Figure 5).92–95

Within the chamber myocardium, each of the four chambers is marked by unique gene

expression patterns. The mouse atria and ventricles express myosin light chain isoforms

MLC2a and MLC2v, respectively.96 Both ventricles additionally express the homeobox gene

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Irx4, which activates ventricular myosin heavy chain 1 (MHC1) and suppresses the atrial

MHC1 isoform in chick.97 In both mouse and chick, Tbx5 expression is absent in the right

ventricle.86 In chick, the right ventricle is also distinguished by Tbx20 expression, which the

left ventricle lacks.35 The basic helix-loop-helix transcription factors eHAND and dHAND

are expressed in the systemic and pulmonary ventricles, respectively, even in situs inversus

mouse embryos.98 In atrial development, high expression of the homeobox gene Pitx2 in the

left atrium suppresses Shox2, thereby inhibiting genes associated with sinoatrial node

development and preventing ectopic pacemaker specification.99

Trabeculation of the Ventricular Myocardium

Trabeculation and subsequent compaction of the ventricular myocardium facilitate septation,

confer greater contractility and conductivity, and help establish the coronary circulation

system in the developing heart.100–104 Paracrine signaling between the endocardium and the

maturing myocardium is an essential driver for the formation of the organized muscular

ridges lining the ventricular wall (Figure 6), a process initiated towards the end of cardiac

looping.105

Central to endocardium-to-myocardium regulation of trabeculation is the transmembrane

receptor Notch1.101, 106 When the extracellular domain of Notch1 binds to ligands such as

Delta4 and Jagged, proteolytic cleavage releases the intracellular domain (N1ICD) into the

cytoplasm.107 N1ICD then associates with its transcriptional cofactor RBPJK and

translocates to the nucleus108, 109 N1ICD/RBPJk upregulates myocardial expression of

BMP10, which stimulates proliferation by decreasing the activity of cell cycle inhibitor

P57kip2 and by up-regulating Tbx20 and Hey2 activity.110, 111 Since Notch1 is

predominantly expressed in endocardial cells lining the base of trabeculae at E9.0, Notch

activity therefore promotes growth of the trabecular myocardium.101 Endothelial-specific

ablation of a N1ICD inhibitor, Fkbp1a, accordingly results in hypertrabeculation.112

Curiously, inactivation of Mib1, an ubiquitinase that promotes ligand binding and

subsequent activation of Notch1, in the myocardium also leads to hypertrabeculation.

Additionally, Mib1-deficient mice have an abnormally thin compact myocardial zone.113

The apparently contradictory effects of Notch1 regulator manipulation suggest that Notch1

plays a complex role in spatiotemporal regulation of trabecular growth.

Notch1 signaling also promotes neuregulin-1 (Nrg1) activity through EphrinB2.101 Nrg1,

one of the endothelial growth factors produced by the endocardium, binds to myocardial

tyrosine kinase receptor ErbB4 and leads to its dimerization with ErbB2, thus activating

signaling cascades modulating cell growth and migration114. Nrg1, ErbB4, and ErbB2 null

mice die in utero around E9.5–10.5 due to embryonic heart failure, with histological

analyses revealing ventricular chambers lacking trabeculae.115–118 Liu et al. have identified

ErbB2 as a dual factor in cardiomyocyte proliferation and directional delamination during

ventricular trabeculation.117 Their assessment of the cardiac function in ErbB2 mutants

revealed decreased ventricular cardiac contractility and bradycardia, supporting previous

reports that Nrg1/ErbB2/4 signaling modulating trabeculae formation may play an important

physiological role in cardiac contractility.119 Additionally, high-resolution imaging of

cardiomyocytes in zebrafish embryos suggests that ErbB2 contributes to robust formation of

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cardiomyocyte surface protrusions and contacts between non-neighboring cardiomyocytes,

which may be required for initiating trabeculation.120 Intriguingly, EphrinB2 expression is

altered by shear stress in embryonic stem cells in vitro, suggesting that trabeculation may

simultaneously contribute to cardiac contractility while responding to the consequential

increase in blood flow.121 This interdependence between cardiogenesis, blood flow, and

other embryonic processes presents challenges for experimental design.

Growth factor signaling from the myocardium to the endocardium also plays an essential

role in sustaining trabeculation: mice that lack expression of Angiopoietin-1, the primary

ligand produced in the myocardium for the endocardial Tie2 receptor, display ventricular

morphology similar to that of the Neuregulin/ErbB4 mutants with further aberrant

phenotypes such as defective angiogenesis, a less intricately-folded immature endocardium,

and embryonic lethality at E12.5.122 Vascular endothelial growth factor (VEGF) is also

produced in the myocardium and binds to the endocardial receptor Flk1. Flk1-deficient mice

lack some endothelial lineages and therefore do not form trabeculae, causing embryonic

lethality by E9.5.106, 123–125 Conversely, overexpression of VEGF results in an attenuated

compact zone and hypertrabeculation.126

The modes of communication between the endocardium and myocardium may be moderated

or complemented by the cardiac jelly, the extracellular matrix between the two layers.

Beginning at E7.5 in the normal embryo, expression of hyaluronic acid synthase-2 (Has2)

contributes to formation of the extracellular matrix, while global Has2 deficiency manifests

as compact ventricular walls lacking trabeculation.127 A potential mechanism through which

Has2 regulates trabeculation is offered by the example of Has2 activation of ErbB2/3 in

cushion mesenchyme formation.128 In later stages of normal cardiac development, the

metalloproteinase ADAMTS1 is highly expressed during E12.5–14.5.129 During this time,

ADAMTS1 eliminates cardiac jelly by cleaving versican, an extracellular matrix

proteoglycan.129, 130 Since versican appears to promote trabecular proliferation, ADAMTS1

expression thereby signals the conclusion of trabecular growth.130 Based on investigations

of similar versican cleavage activity in other embryonic processes, versican proteolysis not

only disrupts functions served by intact versican but may also unmask distinct

morphogenetic functions of the resulting fragments.131, 132 Cleavage of versican by

ADAMTS1 is facilitated by fibulin-1, another extracellular matrix protein that binds both

ADAMTS1 and versican, and global deletion of ADAMTS1 or fibulin-1 results in

hypertrabeculation.129, 130 Endocardial-specific loss around E9.0 of Brg1, an ADAMTS1

inhibitor, allows ADAMTS1 to prematurely thin the cardiac jelly and terminates

trabeculation.129

The development of the cardiac conduction system provides an additional example of

cardiac jelly-mediated endocardium-to-myocardium signaling in relation to trabeculation.

Recent work suggests that endocardial signals promote Cx40 expression in the myocardium

and that the cardiac jelly reduces the intensity of these molecular signals. This model is

supported by the observation that the atrioventricular junction, where the endocardium and

myocardium are separated by relatively thick cardiac jelly during valve formation, has low

Cx40 expression and delayed conduction through the atrioventricular node.133 Endocardial

promotion of connexin expression may also explain why the peripheral ventricular

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conduction system, marked by Cx40 in the mature heart, forms from trabecular

cardiomyocytes closest to the endocardium.134

To further support the interdependence of trabeculation and cardiac conduction system

development, three recent reports have identified mutations in either ion channel HCN4 or

ryanodine receptor RYR2 in comorbid cases of left ventricular non-compaction and certain

familial arrhythmias.135–137 Whereas earlier surveys of LVNC patients suggested that the

non-compact phenotype was arrhythmogenic, these reports motivate further investigation

into the mechanisms by which HCN4 and RYR2, genes previously associated with

arrhythmic disorders, may also perturb myocardial structure.138

Epigenetic Regulation of Heart Development

Acting in concert with signaling pathways and transcription factors, several epigenetic

factors play a critical role in modulating cardiac lineage specification and cardiac

morphogenesis during development. Included in the category of epigenetic regulators are

histone modifications, ATP-dependent chromatin remodeling complexes and DNA

methylation. Each of these regulatory modes influences gene expression by modulating the

accessibility of regulatory DNA sequences to DNA binding proteins, such as transcription

factors. As a result, epigenetic regulation adds an additional layer of complexity to the tight

temporal and spatial control of cardiac gene expression during development.

Covalent modifications to histone proteins alter DNA-histone interactions, resulting in the

establishment of looser or more accessible versus tighter or more restricted chromatin.

Histone acetylation primarily promotes chromatin accessibility, thereby leading to

transcriptional activation. The specific role of histone acetyltransferases (HAT) in heart

development is still unclear. However, mice who carry a global loss-of-function mutation in

the HAT gene p300 die between E12.5 and E15.5, possibly due to the development of

severe heart defects.139 Interestingly, this effect may be due to acetylation and activation of

Gata4 by p300.140 In addition, mice globally lacking the HAT Moz show aortic arch

abnormalities and ventricular septal defects similar to those found in 22q11 deletion

syndrome due to decreased acetylation and therefore transcription of Tbx1.141, 142 In

contrast to HATs, histone deacetylases (HDACs) remove acetyl groups from histones,

resulting in formation of condensed chromatin and transcriptional repression. Cardiac-

specific loss of either Hdac1 or Hdac2 has no significant effect while double mutants

lacking both Hdac1 and Hdac2 in myocardium die in the neonatal period due to cardiac

arrhythmias and dilated cardiomyopathy.143 The deacetylase activity of these epigenetic

factors may not be limited to histone modification. For example, Hdac2 is thought to limit

myocardial proliferation via a partnership with Hopx (homeodomain-only protein) that

results in deacetylation of Gata4, thereby decreasing its transcriptional activity.144, 145

Similarly, Hdac3 may suppress Tbx5 activity by altering acetylation of Tbx5 residues.146

In contrast to histone acetylation, the effect of histone methylation is highly residue-specific,

with methylation of histone H3 at lysine 4, 36 and 79 leading to gene activation while

methylation at lysine 9 and 27 generally results in gene silencing. Both histone

methyltransferases and demethylases have been shown to play critical roles in cardiac

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development, and de novo mutations in genes associated with regulation of histone 3 lysine

4 and lysine 27 methylation have been detected in excess in children with non-familial

congenital heart disease.147 Loss of the histone methyltransferase Smyd1 results in right

ventricular hypoplasia, possibly due to diminished expression of key cardiac transcription

factors Hand2 and Irx4.148 Jarid2, a member of the Jumonji family of histone demethylases,

is a key regulator of cardiac trabeculation (discussed in the preceding section) via repression

of endocardial Notch and Nrg1.149

ATP-dependent chromatin remodeling complexes harness energy from ATP hydrolysis to

alter nucleosome packaging, and thus DNA accessibility.140 Of the four families of ATP-

dependent chromatin remodelers, the family best studied in cardiac development is the

switching defective/sucrose non-fermenting (SWI/SNF) family. The Brg1/BRM-associated

factor (BAF) complex contains twelve subunits, including the ATPase subunit encoded by

either brahma (Brm) or brahma-related gene 1 (Brg1).150 Brg1 has been shown to

genetically interact with several cardiac transcription factors, including Nkx2-5, Gata4, Tbx5

and Tbx20.151, 152 Mice that are globally haploinsufficient for Brg1 show a wide range of

mild cardiac defects, including mild septal defects and aberrant conduction. Interestingly,

mice with both haploinsufficiency for Brg1 as well as haploinsufficiency for Nkx2-5, Tbx5

or Tbx20 show more severe and often lethal cardiac defects than single mutants

alone.153–155 Brg1 also promotes myocardial proliferation through stimulation of Bmp10,

and the thin myocardium phenotype observed in the absence of Brg1 can be rescued with

Bmp10.156 Also of note, murine Brg1 activates fetal beta-myosin heavy chain while

repressing adult alpha-myosin heavy chain, thereby maintaining a fetal phenotype during

development.156 Finally, perhaps the most striking evidence demonstrating a central role for

chromatin remodeling in cardiomyocyte lineage specification is the observation that

overexpression of Tbx5, Gata4, and the BAF subunit Baf60c can promote

transdifferentiation of non-cardiac mesoderm into cardiac tissue.157

DNA methylation involves the addition of a methyl group to cytosine in so-called CpG

islands. Notably, DNA methylation patterns are wiped clean at the time of conception and

are gradually re-introduced during embryogenesis. Recent genome-wide analysis of DNA

methylation patterns in developing mouse hearts demonstrated overall stability in the

amount of DNA methylation between E11.5 and E14.5.158 However, less than 1% of

methylation sites did show differential methylation patterns between the two time-points, of

which about two-thirds were more highly methylated at E14.5.158 Cardiac regulatory genes

were highly enriched in this subset, including signaling molecules such as Wnt2 and Fgf2 as

well as transcription factors such as Gata6 and Mef2c.158 Among those sites with the

greatest amount of differential methylation, half also showed corresponding changes in

expression of the associated gene.158

Summary

As our understanding of the molecular regulation of cardiac development continues to

improve, so will our ability to clarify the determinants of congenital heart disease as well as

our capacity to generate cells for regenerative medicine therapies. In recent years, the

identification of key transcription factors and their requirement in distinct stages of

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cardiomyogenesis has helped lay the groundwork for determining the precise molecular

mechanism involved in commitment of cardiac progenitor cells from their mesodermal

precursors, chamber-specific cardiomyocytes from heart field progenitors, and trabecular

versus compact myocardium from chamber myocytes. Furthermore, the elucidation of

critical epigenetic modifiers that regulate expression of cardiac genes in a spatiotemporal

manner has helped reveal essential mechanisms that modify chromatin at specific locations

for transcription factors to execute their designated roles during development.

Future Perspectives

In order to realize the tremendous potential made by our cumulative investment in cardiac

development biology in recent decades to identify the pathogenic mechanisms of congenital

heart diseases such as hypoplastic left heart syndrome, transposition of great vessels, double

outlet right ventricle, and ventricular non-compaction, further clarification is needed on a

number of key unresolved issues. For example, with regards to the understanding of heart

field precursor commitment, we need greater insight into the differences in epigenetic

regulation between FHF and SHF cells as they may explain the differences in the response

to failure between the right and left ventricle. Specifically, the lack of requirement for Isl1

expression in FHF cells and the differential effects of FGF and BMP signaling in SHF cells

suggest that the biology of right ventricular cardiomyocytes at the single cell level may be

very different from those in the left ventricle.

In the formation of chamber-specific cardiomyocytes, the distinct expression of T-box and

Hand transcription factors along the linear and looping heart tube suggest differences in

epigenetic regulation between atrial versus ventricular, as well as right versus left ventricular

myocyte development. It would be of great value to elucidate the gene expression and

epigenetic profile of all chamber myocyte precursors during the linear heart tube stage of

development at the single cell level. This level of resolution could help us determine the key

regulators at the earliest steps of chamber myocyte formation and their link to chamber-

specific malformations.

Finally, in the context of trabecular versus compact myocardium formation for chamber-

specific myocytes, we need a greater understanding of the role of specific signaling pathway

and biomechanical influences on myocardial maturation. Identifying the answers to these

questions may hold the key to our ability to accurately predict and diagnose congenital heart

diseases and treat them in utero using novel interventional or molecular therapies. Indeed,

one of the most significant barriers to translating pluripotent stem cell (PSC)-derived

cardiomyocytes into regenerative therapies is the inability of these cells to reach full

maturity both in vitro and in vivo after transplantation. Despite significant efforts to enhance

the maturation of these cells by pacing, stretching, and co-culturing with non-myocytes, they

remain largely immature. A better understanding of the key factors that regulate normal

cardiomyocyte maturation during embryonic development should help guide, conceptually,

the designing of new approaches to enhance the maturation of PSC-derived cardiomyocytes

in vitro. If we are successful in this endeavor, the potential for screening and discovering

new drugs using PSC-base disease models to treat defective signaling pathways in

congenital heart disease could be significantly increased.

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In summary, we believe that while major strides have been made in recent years to unravel

the molecular underpinnings of cardiomyogenic lineage commitment, expansion, and

maturation, there remain significant gaps in our knowledge that await further investigation.

With the continuous advancements in stem cell and genome-editing technology, as well as

detailed mechanistic analysis using model organisms, we shall be able to identify and

pinpoint key regulators of cardiomyogenesis and devise approaches to correct their

functional deficiency prior to the development of disease.

Acknowledgments

We apologize to colleagues whose work we could not cite in this brief review article. We thank Dr. Anthony Sturzu for manuscript critique. This article is funded in part by the NIH/NHLBI Progenitor Cell Biology Consortium (U01HL009976-05), NIH Director’s Pioneer Award (DP1LM012179-01), the California Institute for Regenerative Medicine (RB3-05129), the American Heart Association (14GRNT18630016), and an Endowed Faculty Scholar Award from the Child Health Research Institute at Stanford (to S.M.W.)

Non-standard abbreviations and acronyms

Mesp1 Mesoderm Posterior 1

BMP bone morphogenetic protein

FGF fibroblast growth factor

FHF first heart field

SHF second heart field

AV atrioventricular

LIM Lin11, Isl1, Mec3

Isl1 Islet1

ES embryonic stem

DGS DiGeorge syndrome

VCFS velocardiofacial syndrome

VCAM-1 vascular adhesion molecule 1

Cx connexin

MHC myosin heavy chain

N1ICD Notch1 intracellular domain

Nrg1 neuregulin-1

VEGF vascular endothelial growth factor

Has2 hyaluronic acid synthase-2

HAT histone acetyltransferase

HDAC histone deacetylase

Hopx homeodomain-only protein

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SWI/SNF switching defective/sucrose non-fermenting

BAF Brg1/BRM-associated factor

Brm brahma

Brg1 brahma-related gene 1

Shh sonic hedgehog

AVC atrioventricular canal

p-OFT proximal outflow tract

d-OFT distal outflow tract

References

1. Evans SM, Yelon D, Conlon FL, Kirby ML. Myocardial lineage development. Circ Res. 2010; 107:1428–1444. [PubMed: 21148449]

2. Vincent SD, Buckingham ME. How to make a heart: The origin and regulation of cardiac progenitor cells. Curr Top Dev Biol. 2010; 90:1–41. [PubMed: 20691846]

3. Kuo CT, Morrisey EE, Anandappa R, Sigrist K, Lu MM, Parmacek MS, Soudais C, Leiden JM. Gata4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev. 1997; 11:1048–1060. [PubMed: 9136932]

4. Moorman AF, Christoffels VM. Cardiac chamber formation: Development, genes, and evolution. Physiol Rev. 2003; 83:1223–1267. [PubMed: 14506305]

5. Cho CH, Kim SS, Jeong MJ, Lee CO, Shin HS. The na+ -ca2+ exchanger is essential for embryonic heart development in mice. Mol Cells. 2000; 10:712–722. [PubMed: 11211878]

6. Wakimoto K, Kobayashi K, Kuro-O M, Yao A, Iwamoto T, Yanaka N, Kita S, Nishida A, Azuma S, Toyoda Y, Omori K, Imahie H, Oka T, Kudoh S, Kohmoto O, Yazaki Y, Shigekawa M, Imai Y, Nabeshima Y, Komuro I. Targeted disruption of na+/ca2+ exchanger gene leads to cardiomyocyte apoptosis and defects in heartbeat. J Biol Chem. 2000; 275:36991–36998. [PubMed: 10967099]

7. North TE, Goessling W, Peeters M, Li P, Ceol C, Lord AM, Weber GJ, Harris J, Cutting CC, Huang P, Dzierzak E, Zon LI. Hematopoietic stem cell development is dependent on blood flow. Cell. 2009; 137:736–748. [PubMed: 19450519]

8. Reckova M, Rosengarten C, deAlmeida A, Stanley CP, Wessels A, Gourdie RG, Thompson RP, Sedmera D. Hemodynamics is a key epigenetic factor in development of the cardiac conduction system. Circ Res. 2003; 93:77–85. [PubMed: 12775585]

9. Hove JR, Köster RW, Forouhar AS, Acevedo-Bolton G, Fraser SE, Gharib M. Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis. Nature. 2003; 421:172–177. [PubMed: 12520305]

10. Harvey RP. Patterning the vertebrate heart. Nat Rev Genet. 2002; 3:544–556. [PubMed: 12094232]

11. Costello I, Pimeisl IM, Dräger S, Bikoff EK, Robertson EJ, Arnold SJ. The t-box transcription factor eomesodermin acts upstream of mesp1 to specify cardiac mesoderm during mouse gastrulation. Nat Cell Biol. 2011; 13:1084–1091. [PubMed: 21822279]

12. van den Ameele J, Tiberi L, Bondue A, Paulissen C, Herpoel A, Iacovino M, Kyba M, Blanpain C, Vanderhaeghen P. Eomesodermin induces mesp1 expression and cardiac differentiation from embryonic stem cells in the absence of activin. EMBO Rep. 2012; 13:355–362. [PubMed: 22402664]

13. Saga Y, Kitajima S, Miyagawa-Tomita S. Mesp1 expression is the earliest sign of cardiovascular development. Trends Cardiovasc Med. 2000; 10:345–352. [PubMed: 11369261]

14. Bondue A, Lapouge G, Paulissen C, Semeraro C, Iacovino M, Kyba M, Blanpain C. Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification. Cell Stem Cell. 2008; 3:69–84. [PubMed: 18593560]

Paige et al.


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

15. Schultheiss TM, Burch JB, Lassar AB. A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes Dev. 1997; 11:451–462. [PubMed: 9042859]

16. Andrée B, Duprez D, Vorbusch B, Arnold HH, Brand T. Bmp-2 induces ectopic expression of cardiac lineage markers and interferes with somite formation in chicken embryos. Mech Dev. 1998; 70:119–131. [PubMed: 9510029]

17. Pandur P, Lasche M, Eisenberg LM, Kuhl M. Wnt-11 activation of a non-canonical wnt signalling pathway is required for cardiogenesis. Nature. 2002; 418:636–641. [PubMed: 12167861]

18. Tzahor E. Wnt/beta-catenin signaling and cardiogenesis: Timing does matter. Dev Cell. 2007; 13:10–13. [PubMed: 17609106]

19. Marvin MJ, Di Rocco G, Gardiner A, Bush SM, Lassar AB. Inhibition of wnt activity induces heart formation from posterior mesoderm. Genes Dev. 2001; 15:316–327. [PubMed: 11159912]

20. Schneider VA, Mercola M. Wnt antagonism initiates cardiogenesis in xenopus laevis. Genes Dev. 2001; 15:304–315. [PubMed: 11159911]

21. Lickert H, Kutsch S, Kanzler B, Tamai Y, Taketo MM, Kemler R. Formation of multiple hearts in mice following deletion of beta-catenin in the embryonic endoderm. Dev Cell. 2002; 3:171–181. [PubMed: 12194849]

22. Foley AC, Mercola M. Heart induction by wnt antagonists depends on the homeodomain transcription factor hex. Genes Dev. 2005; 19:387–396. [PubMed: 15687261]

23. Kelly RG, Brown NA, Buckingham ME. The arterial pole of the mouse heart forms from fgf10-expressing cells in pharyngeal mesoderm. Dev Cell. 2001; 1:435–440. [PubMed: 11702954]

24. Waldo KL, Kumiski DH, Wallis KT, Stadt HA, Hutson MR, Platt DH, Kirby ML. Conotruncal myocardium arises from a secondary heart field. Development. 2001; 128:3179–3188. [PubMed: 11688566]

25. Mjaatvedt CH, Nakaoka T, Moreno-Rodriguez R, Norris RA, Kern MJ, Eisenberg CA, Turner D, Markwald RR. The outflow tract of the heart is recruited from a novel heart-forming field. Dev Biol. 2001; 238:97–109. [PubMed: 11783996]

26. Lescroart F, Chabab S, Lin X, Rulands S, Paulissen C, Rodolosse A, Auer H, Achouri Y, Dubois C, Bondue A, Simons BD, Blanpain C. Early lineage restriction in temporally distinct populations of mesp1 progenitors during mammalian heart development. Nat Cell Biol. 2014; 16:829–840. [PubMed: 25150979]

27. Devine WP, Wythe JD, George M, Koshiba-Takeuchi K, Bruneau BG. Early patterning and specification of cardiac progenitors in gastrulating mesoderm. Elife. 2014; 3

28. Buckingham M, Meilhac S, Zaffran S. Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet. 2005; 6:826–835. [PubMed: 16304598]

29. Waldo KL, Hutson MR, Ward CC, Zdanowicz M, Stadt HA, Kumiski D, Abu-Issa R, Kirby ML. Secondary heart field contributes myocardium and smooth muscle to the arterial pole of the developing heart. Dev Biol. 2005; 281:78–90. [PubMed: 15848390]

30. Zaffran S, Kelly RG, Meilhac SM, Buckingham ME, Brown NA. Right ventricular myocardium derives from the anterior heart field. Circ Res. 2004; 95:261–268. [PubMed: 15217909]

31. Cai CL, Liang X, Shi Y, Chu PH, Pfaff SL, Chen J, Evans S. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev Cell. 2003; 5:877–889. [PubMed: 14667410]

32. Verzi MP, McCulley DJ, De Val S, Dodou E, Black BL. The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field. Dev Biol. 2005; 287:134–145. [PubMed: 16188249]

33. Kelly RG. The second heart field. Curr Top Dev Biol. 2012; 100:33–65. [PubMed: 22449840]

34. Rochais F, Mesbah K, Kelly RG. Signaling pathways controlling second heart field development. Circ Res. 2009; 104:933–942. [PubMed: 19390062]

35. Takeuchi JK, Ohgi M, Koshiba-Takeuchi K, Shiratori H, Sakaki I, Ogura K, Saijoh Y, Ogura T. Tbx5 specifies the left/right ventricles and ventricular septum position during cardiogenesis. Development. 2003; 130:5953–5964. [PubMed: 14573514]

36. Stanley EG, Biben C, Elefanty A, Barnett L, Koentgen F, Robb L, Harvey RP. Efficient cre-mediated deletion in cardiac progenitor cells conferred by a 3’utr-ires-cre allele of the homeobox gene nkx2–5. Int J Dev Biol. 2002; 46:431–439. [PubMed: 12141429]

Paige et al.


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

37. Liang X, Wang G, Lin L, Lowe J, Zhang Q, Bu L, Chen Y, Chen J, Sun Y, Evans SM. Hcn4 dynamically marks the first heart field and conduction system precursors. Circ Res. 2013; 113:399–407. [PubMed: 23743334]

38. Später D, Abramczuk MK, Buac K, Zangi L, Stachel MW, Clarke J, Sahara M, Ludwig A, Chien KR. A hcn4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells. Nat Cell Biol. 2013; 15:1098–1106. [PubMed: 23974038]

39. Moretti A, Caron L, Nakano A, Lam JT, Bernshausen A, Chen Y, Qyang Y, Bu L, Sasaki M, Martin-Puig S, Sun Y, Evans SM, Laugwitz KL, Chien KR. Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification. Cell. 2006; 127:1151–1165. [PubMed: 17123592]

40. Rana MS, Theveniau-Ruissy M, De Bono C, Mesbah K, Francou A, Rammah M, Dominguez JN, Roux M, Laforest B, Anderson RH, Mohun TJ, Zaffran S, Christoffels VM, Kelly RG. Tbx1 coordinates addition of posterior second heart field progenitor cells to the arterial and venous poles of the heart. Circ Res. 2014

41. Vincent SD, Mayeuf-Louchart A, Watanabe Y, Brzezinski JA, Miyagawa-Tomita S, Kelly RG, Buckingham M. Prdm1 functions in the mesoderm of the second heart field, where it interacts genetically with tbx1, during outflow tract morphogenesis in the mouse embryo. Hum Mol Genet. 2014

42. Ma HY, Xu J, Eng D, Gross MK, Kioussi C. Pitx2-mediated cardiac outflow tract remodeling. Dev Dyn. 2013; 242:456–468. [PubMed: 23361844]

43. Bu L, Jiang X, Martin-Puig S, Caron L, Zhu S, Shao Y, Roberts DJ, Huang PL, Domian IJ, Chien KR. Human isl1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature. 2009; 460:113–117. [PubMed: 19571884]

44. Laugwitz KL, Moretti A, Lam J, Gruber P, Chen Y, Woodard S, Lin LZ, Cai CL, Lu MM, Reth M, Platoshyn O, Yuan JX, Evans S, Chien KR. Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature. 2005; 433:647–653. [PubMed: 15703750]

45. Weinberger F, Mehrkens D, Friedrich FW, Stubbendorff M, Hua X, Müller JC, Schrepfer S, Evans SM, Carrier L, Eschenhagen T. Localization of islet-1-positive cells in the healthy and infarcted adult murine heart. Circ Res. 2012; 110:1303–1310. [PubMed: 22427341]

46. Park EJ, Ogden LA, Talbot A, Evans S, Cai CL, Black BL, Frank DU, Moon AM. Required, tissue-specific roles for fgf8 in outflow tract formation and remodeling. Development. 2006; 133:2419–2433. [PubMed: 16720879]

47. Watanabe Y, Zaffran S, Kuroiwa A, Higuchi H, Ogura T, Harvey RP, Kelly RG, Buckingham M. Fibroblast growth factor 10 gene regulation in the second heart field by tbx1, nkx2–5, and islet1 reveals a genetic switch for down-regulation in the myocardium. Proc Natl Acad Sci U S A. 2012; 109:18273–18280. [PubMed: 23093675]

48. Zaffran S, Kelly RG. New developments in the second heart field. Differentiation. 2012; 84:17–24. [PubMed: 22521611]

49. Dyer LA, Kirby ML. Sonic hedgehog maintains proliferation in secondary heart field progenitors and is required for normal arterial pole formation. Dev Biol. 2009; 330:305–317. [PubMed: 19361493]

50. Yamagishi H, Maeda J, Hu T, McAnally J, Conway SJ, Kume T, Meyers EN, Yamagishi C, Srivastava D. Tbx1 is regulated by tissue-specific forkhead proteins through a common sonic hedgehog-responsive enhancer. Genes Dev. 2003; 17:269–281. [PubMed: 12533514]

51. Seo S, Kume T. Forkhead transcription factors, foxc1 and foxc2, are required for the morphogenesis of the cardiac outflow tract. Dev Biol. 2006; 296:421–436. [PubMed: 16839542]

52. Aggarwal VS, Liao J, Bondarev A, Schimmang T, Lewandoski M, Locker J, Shanske A, Campione M, Morrow BE. Dissection of tbx1 and fgf interactions in mouse models of 22q11ds suggests functional redundancy. Hum Mol Genet. 2006; 15:3219–3228. [PubMed: 17000704]

53. Pane LS, Zhang Z, Ferrentino R, Huynh T, Cutillo L, Baldini A. Tbx1 is a negative modulator of mef2c. Hum Mol Genet. 2012; 21:2485–2496. [PubMed: 22367967]

54. Chen L, Fulcoli FG, Tang S, Baldini A. Tbx1 regulates proliferation and differentiation of multipotent heart progenitors. Circ Res. 2009; 105:842–851. [PubMed: 19745164]

Paige et al.


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

55. Lin L, Cui L, Zhou W, Dufort D, Zhang X, Cai CL, Bu L, Yang L, Martin J, Kemler R, Rosenfeld MG, Chen J, Evans SM. Beta-catenin directly regulates islet1 expression in cardiovascular progenitors and is required for multiple aspects of cardiogenesis. Proc Natl Acad Sci U S A. 2007; 104:9313–9318. [PubMed: 17519333]

56. Kirby ML, Hutson MR. Factors controlling cardiac neural crest cell migration. Cell Adh Migr. 2010; 4

57. Hutson MR, Kirby ML. Model systems for the study of heart development and disease. Cardiac neural crest and conotruncal malformations. Semin Cell Dev Biol. 2007; 18:101–110. [PubMed: 17224285]

58. Arima Y, Miyagawa-Tomita S, Maeda K, Asai R, Seya D, Minoux M, Rijli FM, Nishiyama K, Kim KS, Uchijima Y, Ogawa H, Kurihara Y, Kurihara H. Preotic neural crest cells contribute to coronary artery smooth muscle involving endothelin signalling. Nat Commun. 2012; 3:1267. [PubMed: 23232397]

59. Escot S, Blavet C, Härtle S, Duband JL, Fournier-Thibault C. Misregulation of sdf1-cxcr4 signaling impairs early cardiac neural crest cell migration leading to conotruncal defects. Circ Res. 2013; 113:505–516. [PubMed: 23838132]

60. Holler KL, Hendershot TJ, Troy SE, Vincentz JW, Firulli AB, Howard MJ. Targeted deletion of hand2 in cardiac neural crest-derived cells influences cardiac gene expression and outflow tract development. Dev Biol. 2010; 341:291–304. [PubMed: 20144608]

61. Farrell MJ, Burch JL, Wallis K, Rowley L, Kumiski D, Stadt H, Godt RE, Creazzo TL, Kirby ML. Fgf-8 in the ventral pharynx alters development of myocardial calcium transients after neural crest ablation. J Clin Invest. 2001; 107:1509–1517. [PubMed: 11413158]

62. Keyte A, Hutson MR. The neural crest in cardiac congenital anomalies. Differentiation. 2012; 84:25–40. [PubMed: 22595346]

63. High FA, Jain R, Stoller JZ, Antonucci NB, Lu MM, Loomes KM, Kaestner KH, Pear WS, Epstein JA. Murine jagged1/notch signaling in the second heart field orchestrates fgf8 expression and tissue-tissue interactions during outflow tract development. J Clin Invest. 2009; 119:1986–1996. [PubMed: 19509466]

64. de la Pompa JL, Epstein JA. Coordinating tissue interactions: Notch signaling in cardiac development and disease. Dev Cell. 2012; 22:244–254. [PubMed: 22340493]

65. Yang JT, Rayburn H, Hynes RO. Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development. Development. 1995; 121:549–560. [PubMed: 7539359]

66. Kwee L, Baldwin HS, Shen HM, Stewart CL, Buck C, Buck CA, Labow MA. Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (vcam-1) deficient mice. Development. 1995; 121:489–503. [PubMed: 7539357]

67. del Monte G, Casanova JC, Guadix JA, MacGrogan D, Burch JB, Pérez-Pomares JM, de la Pompa JL. Differential notch signaling in the epicardium is required for cardiac inflow development and coronary vessel morphogenesis. Circ Res. 2011; 108:824–836. [PubMed: 21311046]

68. Olivey HE, Compton LA, Barnett JV. Coronary vessel development: The epicardium delivers. Trends Cardiovasc Med. 2004; 14:247–251. [PubMed: 15451517]

69. Red-Horse K, Ueno H, Weissman IL, Krasnow MA. Coronary arteries form by developmental reprogramming of venous cells. Nature. 2010; 464:549–553. [PubMed: 20336138]

70. Katz TC, Singh MK, Degenhardt K, Rivera-Feliciano J, Johnson RL, Epstein JA, Tabin CJ. Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells. Dev Cell. 2012; 22:639–650. [PubMed: 22421048]

71. Zhou B, Ma Q, Rajagopal S, Wu SM, Domian I, Rivera-Feliciano J, Jiang D, von Gise A, Ikeda S, Chien KR, Pu WT. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature. 2008; 454:109–113. [PubMed: 18568026]

72. Cai CL, Martin JC, Sun Y, Cui L, Wang L, Ouyang K, Yang L, Bu L, Liang X, Zhang X, Stallcup WB, Denton CP, McCulloch A, Chen J, Evans SM. A myocardial lineage derives from tbx18 epicardial cells. Nature. 2008; 454:104–108. [PubMed: 18480752]

73. Masters M, Riley PR. The epicardium signals the way towards heart regeneration. Stem Cell Res. 2014

Paige et al.


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

74. Nakajima Y, Imanaka-Yoshida K. New insights into the developmental mechanisms of coronary vessels and epicardium. Int Rev Cell Mol Biol. 2013; 303:263–317. [PubMed: 23445813]

75. Ruiz-Villalba A, Pérez-Pomares JM. The expanding role of the epicardium and epicardial-derived cells in cardiac development and disease. Curr Opin Pediatr. 2012; 24:569–576. [PubMed: 22890066]

76. Pérez-Pomares JM, de la Pompa JL. Signaling during epicardium and coronary vessel development. Circ Res. 2011; 109:1429–1442. [PubMed: 22158650]

77. Soufan AT, van den Berg G, Ruijter JM, de Boer PA, van den Hoff MJ, Moorman AF. Regionalized sequence of myocardial cell growth and proliferation characterizes early chamber formation. Circ Res. 2006; 99:545–552. [PubMed: 16888243]

78. Rana MS, Christoffels VM, Moorman AF. A molecular and genetic outline of cardiac morphogenesis. Acta Physiol (Oxf). 2013; 207:588–615. [PubMed: 23297764]

79. Lin YF, Swinburne I, Yelon D. Multiple influences of blood flow on cardiomyocyte hypertrophy in the embryonic zebrafish heart. Dev Biol. 2012; 362:242–253. [PubMed: 22192888]

80. Dietrich AC, Lombardo VA, Abdelilah-Seyfried S. Blood flow and bmp signaling control endocardial chamber morphogenesis. Dev Cell. 2014; 30:367–377. [PubMed: 25158852]

81. Auman HJ, Coleman H, Riley HE, Olale F, Tsai HJ, Yelon D. Functional modulation of cardiac form through regionally confined cell shape changes. PLoS Biol. 2007; 5:e53. [PubMed: 17311471]

82. Christoffels VM, Smits GJ, Kispert A, Moorman AF. Development of the pacemaker tissues of the heart. Circ Res. 2010; 106:240–254. [PubMed: 20133910]

83. Hoogaars WM, Barnett P, Moorman AF, Christoffels VM. T-box factors determine cardiac design. Cell Mol Life Sci. 2007; 64:646–660. [PubMed: 17380306]

84. Moskowitz IP, Pizard A, Patel VV, Bruneau BG, Kim JB, Kupershmidt S, Roden D, Berul CI, Seidman CE, Seidman JG. The t-box transcription factor tbx5 is required for the patterning and maturation of the murine cardiac conduction system. Development. 2004; 131:4107–4116. [PubMed: 15289437]

85. Hatcher CJ, Basson CT. Specification of the cardiac conduction system by transcription factors. Circ Res. 2009; 105:620–630. [PubMed: 19797194]

86. Bruneau BG, Logan M, Davis N, Levi T, Tabin CJ, Seidman JG, Seidman CE. Chamber-specific cardiac expression of tbx5 and heart defects in holt-oram syndrome. Dev Biol. 1999; 211:100–108. [PubMed: 10373308]

87. Christoffels VM, Hoogaars WM, Tessari A, Clout DE, Moorman AF, Campione M. T-box transcription factor tbx2 represses differentiation and formation of the cardiac chambers. Dev Dyn. 2004; 229:763–770. [PubMed: 15042700]

88. Singh R, Hoogaars WM, Barnett P, Grieskamp T, Rana MS, Buermans H, Farin HF, Petry M, Heallen T, Martin JF, Moorman AF, ‘t Hoen PA, Kispert A, Christoffels VM. Tbx2 and tbx3 induce atrioventricular myocardial development and endocardial cushion formation. Cell Mol Life Sci. 2012; 69:1377–1389. [PubMed: 22130515]

89. Mommersteeg MT, Hoogaars WM, Prall OW, de Gier-de Vries C, Wiese C, Clout DE, Papaioannou VE, Brown NA, Harvey RP, Moorman AF, Christoffels VM. Molecular pathway for the localized formation of the sinoatrial node. Circ Res. 2007; 100:354–362. [PubMed: 17234970]

90. Liberatore CM, Searcy-Schrick RD, Yutzey KE. Ventricular expression of tbx5 inhibits normal heart chamber development. Dev Biol. 2000; 223:169–180. [PubMed: 10864469]

91. Habets PE, Moorman AF, Clout DE, van Roon MA, Lingbeek M, van Lohuizen M, Campione M, Christoffels VM. Cooperative action of tbx2 and nkx2.5 inhibits anf expression in the atrioventricular canal: Implications for cardiac chamber formation. Genes Dev. 2002; 16:1234–1246. [PubMed: 12023302]

92. Cai CL, Zhou W, Yang L, Bu L, Qyang Y, Zhang X, Li X, Rosenfeld MG, Chen J, Evans S. T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis. Development. 2005; 132:2475–2487. [PubMed: 15843407]

93. Singh MK, Christoffels VM, Dias JM, Trowe MO, Petry M, Schuster-Gossler K, Bürger A, Ericson J, Kispert A. Tbx20 is essential for cardiac chamber differentiation and repression of tbx2. Development. 2005; 132:2697–2707. [PubMed: 15901664]

Paige et al.


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

94. Stennard FA, Harvey RP. T-box transcription factors and their roles in regulatory hierarchies in the developing heart. Development. 2005; 132:4897–4910. [PubMed: 16258075]

95. Takeuchi JK, Mileikovskaia M, Koshiba-Takeuchi K, Heidt AB, Mori AD, Arruda EP, Gertsenstein M, Georges R, Davidson L, Mo R, Hui CC, Henkelman RM, Nemer M, Black BL, Nagy A, Bruneau BG. Tbx20 dose-dependently regulates transcription factor networks required for mouse heart and motoneuron development. Development. 2005; 132:2463–2474. [PubMed: 15843409]

96. Small EM, Krieg PA. Molecular regulation of cardiac chamber-specific gene expression. Trends Cardiovasc Med. 2004; 14:13–18. [PubMed: 14720469]

97. Bao ZZ, Bruneau BG, Seidman JG, Seidman CE, Cepko CL. Regulation of chamber-specific gene expression in the developing heart by irx4. Science. 1999; 283:1161–1164. [PubMed: 10024241]

98. Thomas T, Yamagishi H, Overbeek PA, Olson EN, Srivastava D. The bhlh factors, dhand and ehand, specify pulmonary and systemic cardiac ventricles independent of left-right sidedness. Dev Biol. 1998; 196:228–236. [PubMed: 9576835]

99. Wang J, Klysik E, Sood S, Johnson RL, Wehrens XH, Martin JF. Pitx2 prevents susceptibility to atrial arrhythmias by inhibiting left-sided pacemaker specification. Proc Natl Acad Sci U S A. 2010; 107:9753–9758. [PubMed: 20457925]

100. Ben-Shachar G, Arcilla RA, Lucas RV, Manasek FJ. Ventricular trabeculations in the chick embryo heart and their contribution to ventricular and muscular septal development. Circ Res. 1985; 57:759–766. [PubMed: 4053307]

101. Grego-Bessa J, Luna-Zurita L, del Monte G, Bolos V, Melgar P, Arandilla A, Garratt AN, Zang H, Mukouyama YS, Chen H, Shou W, Ballestar E, Esteller M, Rojas A, Perez-Pomares JM, de la Pompa JL. Notch signaling is essential for ventricular chamber development. Developmental cell. 2007; 12:415–429. [PubMed: 17336907]

102. Rentschler S, Zander J, Meyers K, France D, Levine R, Porter G, Rivkees SA, Morley GE, Fishman GI. Neuregulin-1 promotes formation of the murine cardiac conduction system. Proc Natl Acad Sci U S A. 2002; 99:10464–10469. [PubMed: 12149465]

103. Tian X, Hu T, Zhang H, He L, Huang X, Liu Q, Yu W, He L, Yang Z, Yan Y, Yang X, Zhong TP, Pu WT, Zhou B. Vessel formation. De novo formation of a distinct coronary vascular population in neonatal heart. Science (New York, NY). 2014; 345:90–94.

104. Moorman AF, Christoffels VM. Development of the cardiac conduction system: A matter of chamber development. Novartis Found Symp. 2003; 250:25–34. discussion 34–43, 276–279. [PubMed: 12956322]

105. Moorman AFM, Christoffels VM. Cardiac chamber formation: Development, genes, and evolution. 2003

106. Zhang W, Chen H, Qu X, Chang CP, Shou W. Molecular mechanism of ventricular trabeculation/compaction and the pathogenesis of the left ventricular noncompaction cardiomyopathy (lvnc). Am J Med Genet C Semin Med Genet. 2013; 163C:144–156. [PubMed: 23843320]

107. Kopan R. Notch: A membrane-bound transcription factor. J Cell Sci. 2002; 115:1095–1097. [PubMed: 11884509]

108. Tamura K, Taniguchi Y, Minoguchi S, Sakai T, Tun T, Furukawa T, Honjo T. Physical interaction between a novel domain of the receptor notch and the transcription factor rbp-j kappa/su(h). Curr Biol. 1995; 5:1416–1423. [PubMed: 8749394]

109. Jarriault S, Brou C, Logeat F, Schroeter EH, Kopan R, Israel A. Signalling downstream of activated mammalian notch. Nature. 1995; 377:355–358. [PubMed: 7566092]

110. Chen H, Shi S, Acosta L, Li W, Lu J, Bao S, Chen Z, Yang Z, Schneider MD, Chien KR, Conway SJ, Yoder MC, Haneline LS, Franco D, Shou W. Bmp10 is essential for maintaining cardiac growth during murine cardiogenesis. Development. 2004; 131:2219–2231. [PubMed: 15073151]

111. Zhang W, Chen H, Wang Y, Yong W, Zhu W, Liu Y, Wagner GR, Payne RM, Field LJ, Xin H, Cai CL, Shou W. Tbx20 transcription factor is a downstream mediator for bone morphogenetic protein-10 in regulating cardiac ventricular wall development and function. J Biol Chem. 2011; 286:36820–36829. [PubMed: 21890625]

112. Chen H, Zhang W, Sun X, Yoshimoto M, Chen Z, Zhu W, Liu J, Shen Y, Yong W, Li D, Zhang J, Lin Y, Li B, VanDusen NJ, Snider P, Schwartz RJ, Conway SJ, Field LJ, Yoder MC, Firulli

Paige et al.


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

AB, Carlesso N, Towbin JA, Shou W. Fkbp1a controls ventricular myocardium trabeculation and compaction by regulating endocardial notch1 activity. Development. 2013; 140:1946–1957. [PubMed: 23571217]

113. Luxán G, Casanova JC, Martínez-Poveda B, Prados B, D’Amato G, MacGrogan D, Gonzalez-Rajal A, Dobarro D, Torroja C, Martinez F, Izquierdo-García JL, Fernández-Friera L, Sabater-Molina M, Kong YY, Pizarro G, Ibañez B, Medrano C, García-Pavía P, Gimeno JR, Monserrat L, Jiménez-Borreguero LJ, de la Pompa JL. Mutations in the notch pathway regulator mib1 cause left ventricular noncompaction cardiomyopathy. Nat Med. 2013; 19:193–201. [PubMed: 23314057]

114. Yarden Y, Sliwkowski MX. Untangling the erbb signalling network. Nat Rev Mol Cell Biol. 2001; 2:127–137. [PubMed: 11252954]

115. Meyer D, Birchmeier C. Multiple essential functions of neuregulin in development. Nature. 1995; 378:386–390. [PubMed: 7477375]

116. Gassmann M, Casagranda F, Orioli D, Simon H, Lai C, Klein R, Lemke G. Aberrant neural and cardiac development in mice lacking the erbb4 neuregulin receptor. Nature. 1995; 378:390–394. [PubMed: 7477376]

117. Liu J, Bressan M, Hassel D, Huisken J, Staudt D, Kikuchi K, Poss KD, Mikawa T, Stainier DY. A dual role for erbb2 signaling in cardiac trabeculation. Development. 2010; 137:3867–3875. [PubMed: 20978078]

118. Chan R, Hardy WR, Laing MA, Hardy SE, Muller WJ. The catalytic activity of the erbb-2 receptor tyrosine kinase is essential for embryonic development. Molecular and cellular biology. 2002; 22:1073–1078. [PubMed: 11809799]

119. Liu X, Gu X, Li Z, Li X, Li H, Chang J, Chen P, Jin J, Xi B, Chen D, Lai D, Graham RM, Zhou M. Neuregulin-1/erbb-activation improves cardiac function and survival in models of ischemic, dilated, and viral cardiomyopathy. Journal of the American College of Cardiology. 2006; 48:1438–1447. [PubMed: 17010808]

120. Staudt DW, Liu J, Thorn KS, Stuurman N, Liebling M, Stainier DY. High-resolution imaging of cardiomyocyte behavior reveals two distinct steps in ventricular trabeculation. Development. 2014; 141:585–593. [PubMed: 24401373]

121. Masumura T, Yamamoto K, Shimizu N, Obi S, Ando J. Shear stress increases expression of the arterial endothelial marker ephrinb2 in murine es cells via the vegf-notch signaling pathways. Arterioscler Thromb Vasc Biol. 2009; 29:2125–2131. [PubMed: 19797707]

122. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD. Requisite role of angiopoietin-1, a ligand for the tie2 receptor, during embryonic angiogenesis. Cell. 1996; 87:1171–1180. [PubMed: 8980224]

123. Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML, Schuh AC. Failure of blood-island formation and vasculogenesis in flk-1-deficient mice. Nature. 1995; 376:62–66. [PubMed: 7596435]

124. Milgrom-Hoffman M, Harrelson Z, Ferrara N, Zelzer E, Evans SM, Tzahor E. The heart endocardium is derived from vascular endothelial progenitors. Development. 2011; 138:4777–4787. [PubMed: 21989917]

125. Tian Y, Morrisey EE. Importance of myocyte-nonmyocyte interactions in cardiac development and disease. Circ Res. 2012; 110:1023–1034. [PubMed: 22461366]

126. Miquerol L, Langille BL, Nagy A. Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression. Development. 2000; 127:3941–3946. [PubMed: 10952892]

127. Camenisch TD, Spicer AP, Brehm-Gibson T, Biesterfeldt J, Augustine ML, Calabro A, Kubalak S, Klewer SE, McDonald JA. Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme. J Clin Invest. 2000; 106:349–360. [PubMed: 10930438]

128. Camenisch TD, Schroeder JA, Bradley J, Klewer SE, McDonald JA. Heart-valve mesenchyme formation is dependent on hyaluronan-augmented activation of erbb2-erbb3 receptors. Nat Med. 2002; 8:850–855. [PubMed: 12134143]

Paige et al.


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

129. Stankunas K, Hang CT, Tsun ZY, Chen H, Lee NV, Wu JI, Shang C, Bayle JH, Shou W, Iruela-Arispe ML, Chang CP. Endocardial brg1 represses adamts1 to maintain the microenvironment for myocardial morphogenesis. Dev Cell. 2008; 14:298–311. [PubMed: 18267097]

130. Cooley MA, Fresco VM, Dorlon ME, Twal WO, Lee NV, Barth JL, Kern CB, Iruela-Arispe ML, Argraves WS. Fibulin-1 is required during cardiac ventricular morphogenesis for versican cleavage, suppression of erbb2 and erk1/2 activation, and to attenuate trabecular cardiomyocyte proliferation. Dev Dyn. 2012; 241:303–314. [PubMed: 22183742]

131. Kern CB, Wessels A, McGarity J, Dixon LJ, Alston E, Argraves WS, Geeting D, Nelson CM, Menick DR, Apte SS. Reduced versican cleavage due to adamts9 haploinsufficiency is associated with cardiac and aortic anomalies. Matrix Biol. 2010; 29:304–316. [PubMed: 20096780]

132. Kern CB, Norris RA, Thompson RP, Argraves WS, Fairey SE, Reyes L, Hoffman S, Markwald RR, Mjaatvedt CH. Versican proteolysis mediates myocardial regression during outflow tract development. Dev Dyn. 2007; 236:671–683. [PubMed: 17226818]

133. Bressan M, Yang PB, Louie JD, Navetta AM, Garriock RJ, Mikawa T. Reciprocal myocardial-endocardial interactions pattern the delay in atrioventricular junction conduction. Development. 2014; 141:4149–4157. [PubMed: 25273084]

134. Christoffels VM, Moorman AF. Development of the cardiac conduction system: Why are some regions of the heart more arrhythmogenic than others? Circ Arrhythm Electrophysiol. 2009; 2:195–207. [PubMed: 19808465]

135. Milano A, Vermeer AM, Lodder EM, Barc J, Verkerk AO, Postma AV, van der Bilt IA, Baars MJ, van Haelst PL, Caliskan K, Hoedemaekers YM, Le Scouarnec S, Redon R, Pinto YM, Christiaans I, Wilde AA, Bezzina CR. Hcn4 mutations in multiple families with bradycardia and left ventricular noncompaction cardiomyopathy. J Am Coll Cardiol. 2014; 64:745–756. [PubMed: 25145517]

136. Schweizer PA, Schröter J, Greiner S, Haas J, Yampolsky P, Mereles D, Buss SJ, Seyler C, Bruehl C, Draguhn A, Koenen M, Meder B, Katus HA, Thomas D. The symptom complex of familial sinus node dysfunction and myocardial noncompaction is associated with mutations in the hcn4 channel. J Am Coll Cardiol. 2014; 64:757–767. [PubMed: 25145518]

137. Ohno S, Omura M, Kawamura M, Kimura H, Itoh H, Makiyama T, Ushinohama H, Makita N, Horie M. Exon 3 deletion of ryr2 encoding cardiac ryanodine receptor is associated with left ventricular non-compaction. Europace. 2014

138. Brescia ST, Rossano JW, Pignatelli R, Jefferies JL, Price JF, Decker JA, Denfield SW, Dreyer WJ, Smith O, Towbin JA, Kim JJ. Mortality and sudden death in pediatric left ventricular noncompaction in a tertiary referral center. Circulation. 2013; 127:2202–2208. [PubMed: 23633270]

139. Shikama N, Lutz W, Kretzschmar R, Sauter N, Roth JF, Marino S, Wittwer J, Scheidweiler A, Eckner R. Essential function of p300 acetyltransferase activity in heart, lung and small intestine formation. EMBO J. 2003; 22:5175–5185. [PubMed: 14517255]

140. Chang CP, Bruneau BG. Epigenetics and cardiovascular development. Annu Rev Physiol. 2012; 74:41–68. [PubMed: 22035349]

141. Voss AK, Vanyai HK, Collin C, Dixon MP, McLennan TJ, Sheikh BN, Scambler P, Thomas T. Moz regulates the tbx1 locus, and moz mutation partially phenocopies digeorge syndrome. Dev Cell. 2012; 23:652–663. [PubMed: 22921202]

142. McElhinney DB, Driscoll DA, Levin ER, Jawad AF, Emanuel BS, Goldmuntz E. Chromosome 22q11 deletion in patients with ventricular septal defect: Frequency and associated cardiovascular anomalies. Pediatrics. 2003; 112:e472. [PubMed: 14654648]

143. Montgomery RL, Davis CA, Potthoff MJ, Haberland M, Fielitz J, Qi X, Hill JA, Richardson JA, Olson EN. Histone deacetylases 1 and 2 redundantly regulate cardiac morphogenesis, growth, and contractility. Genes Dev. 2007; 21:1790–1802. [PubMed: 17639084]

144. Trivedi CM, Zhu W, Wang Q, Jia C, Kee HJ, Li L, Hannenhalli S, Epstein JA. Hopx and hdac2 interact to modulate gata4 acetylation and embryonic cardiac myocyte proliferation. Dev Cell. 2010; 19:450–459. [PubMed: 20833366]

145. Chen F, Kook H, Milewski R, Gitler AD, Lu MM, Li J, Nazarian R, Schnepp R, Jen K, Biben C, Runke G, Mackay JP, Novotny J, Schwartz RJ, Harvey RP, Mullins MC, Epstein JA. Hop is an

Paige et al.


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

unusual homeobox gene that modulates cardiac development. Cell. 2002; 110:713–723. [PubMed: 12297045]

146. Lewandowski SL, Janardhan HP, Smee KM, Bachman M, Sun Z, Lazar MA, Trivedi CM. Histone deacetylase 3 modulates tbx5 activity to regulate early cardiogenesis. Hum Mol Genet. 2014; 23:3801–3809. [PubMed: 24565863]

147. Zaidi S, Choi M, Wakimoto H, Ma L, Jiang J, Overton JD, Romano-Adesman A, Bjornson RD, Breitbart RE, Brown KK, Carriero NJ, Cheung YH, Deanfield J, DePalma S, Fakhro KA, Glessner J, Hakonarson H, Italia MJ, Kaltman JR, Kaski J, Kim R, Kline JK, Lee T, Leipzig J, Lopez A, Mane SM, Mitchell LE, Newburger JW, Parfenov M, Pe’er I, Porter G, Roberts AE, Sachidanandam R, Sanders SJ, Seiden HS, State MW, Subramanian S, Tikhonova IR, Wang W, Warburton D, White PS, Williams IA, Zhao H, Seidman JG, Brueckner M, Chung WK, Gelb BD, Goldmuntz E, Seidman CE, Lifton RP. De novo mutations in histone-modifying genes in congenital heart disease. Nature. 2013; 498:220–223. [PubMed: 23665959]

148. Gottlieb PD, Pierce SA, Sims RJ, Yamagishi H, Weihe EK, Harriss JV, Maika SD, Kuziel WA, King HL, Olson EN, Nakagawa O, Srivastava D. Bop encodes a muscle-restricted protein containing mynd and set domains and is essential for cardiac differentiation and morphogenesis. Nat Genet. 2002; 31:25–32. [PubMed: 11923873]

149. Mysliwiec MR, Bresnick EH, Lee Y. Endothelial jarid2/jumonji is required for normal cardiac development and proper notch1 expression. J Biol Chem. 2011; 286:17193–17204. [PubMed: 21402699]

150. Bevilacqua A, Willis MS, Bultman SJ. Swi/snf chromatin-remodeling complexes in cardiovascular development and disease. Cardiovasc Pathol. 2014; 23:85–91. [PubMed: 24183004]

151. Lickert H, Takeuchi JK, Von Both I, Walls JR, McAuliffe F, Adamson SL, Henkelman RM, Wrana JL, Rossant J, Bruneau BG. Baf60c is essential for function of baf chromatin remodelling complexes in heart development. Nature. 2004; 432:107–112. [PubMed: 15525990]

152. Takeuchi JK, Lou X, Alexander JM, Sugizaki H, Delgado-Olguín P, Holloway AK, Mori AD, Wylie JN, Munson C, Zhu Y, Zhou YQ, Yeh RF, Henkelman RM, Harvey RP, Metzger D, Chambon P, Stainier DY, Pollard KS, Scott IC, Bruneau BG. Chromatin remodelling complex dosage modulates transcription factor function in heart development. Nat Commun. 2011; 2:187. [PubMed: 21304516]

153. Biben C, Weber R, Kesteven S, Stanley E, McDonald L, Elliott DA, Barnett L, Köentgen F, Robb L, Feneley M, Harvey RP. Cardiac septal and valvular dysmorphogenesis in mice heterozygous for mutations in the homeobox gene nkx2-5. Circ Res. 2000; 87:888–895. [PubMed: 11073884]

154. Stennard FA, Costa MW, Lai D, Biben C, Furtado MB, Solloway MJ, McCulley DJ, Leimena C, Preis JI, Dunwoodie SL, Elliott DE, Prall OW, Black BL, Fatkin D, Harvey RP. Murine t-box transcription factor tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation. Development. 2005; 132:2451–2462. [PubMed: 15843414]

155. Nührenberg T, Gilsbach R, Preissl S, Schnick T, Hein L. Epigenetics in cardiac development, function, and disease. Cell Tissue Res. 2014; 356:585–600. [PubMed: 24817102]

156. Hang CT, Yang J, Han P, Cheng HL, Shang C, Ashley E, Zhou B, Chang CP. Chromatin regulation by brg1 underlies heart muscle development and disease. Nature. 2010; 466:62–67. [PubMed: 20596014]

157. Takeuchi JK, Bruneau BG. Directed transdifferentiation of mouse mesoderm to heart tissue by defined factors. Nature. 2009; 459:708–711. [PubMed: 19396158]

158. Chamberlain AA, Lin M, Lister RL, Maslov AA, Wang Y, Suzuki M, Wu B, Greally JM, Zheng D, Zhou B. DNA methylation is developmentally regulated for genes essential for cardiogenesis. J Am Heart Assoc. 2014; 3

159. Bruneau BG. The developmental genetics of congenital heart disease. Nature. 2008; 451:943–948. [PubMed: 18288184]

160. Pérez-Pomares JM, Carmona R, González-Iriarte M, Atencia G, Wessels A, Muñoz-Chápuli R. Origin of coronary endothelial cells from epicardial mesothelium in avian embryos. Int J Dev Biol. 2002; 46:1005–1013. [PubMed: 12533024]

Paige et al.


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161. Wu B, Zhang Z, Lui W, Chen X, Wang Y, Chamberlain AA, Moreno-Rodriguez RA, Markwald RR, O’Rourke BP, Sharp DJ, Zheng D, Lenz J, Baldwin HS, Chang CP, Zhou B. Endocardial cells form the coronary arteries by angiogenesis through myocardial-endocardial vegf signaling. Cell. 2012; 151:1083–1096. [PubMed: 23178125]

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Figure 1. Overview of heart developmentThe cardiac crescent consisting of the first and second heart field cardiac progenitors is

established during late gastrulation. The subsequent proliferation and differentiation of cells

in the first heart field (FHF) leads to the formation of a linear heart tube, giving rise

primarily to the left ventricle and a portion of the atria. The cardiac progenitors in the

anterior second heart field (SHF) contribute to the right ventricle and the outflow tract while

the posterior SHF cells give rise to the atria and the inflow tract.159 Extension and rightward

looping of the linear heart tube allow cranial positioning of the atria with respect to the

ventricles. Remodeling events modulate chamber formation, septation, and valve

development, resulting in formation of the four-chambered heart. Transcription factors that

regulate each stage of heart development are listed. FHF and its derivatives are shown in

orange. SHF and its derivatives are shown in blue.

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Figure 2. Regulation of cardiac mesoderm specificationShown is a cross-section of an E7.5 mouse embryo detailing the signaling pathways that

regulate cardiac specification within splanchnic mesoderm. Factors secreted by the adjacent

endoderm that support cardiac mesoderm specification include FGF, BMP and Shh.

Additionally, non-canonical Wnt ligands, such as Wnt11, expressed in splanchnic mesoderm

also promote cardiac differentiation. Conversely, canonical Wnt ligands, including Wnt1,

Wnt3a and Wnt8, secreted from the overlying neuroectoderm as well as BMP antagonists

Noggin and Chordin secreted from the notochord inhibit cardiac mesoderm specification,

thereby limiting the size of the cardiogenic fields. Abbreviations: FGF, fibroblast growth

factor; BMP, bone morphogenetic protein; Shh, sonic hedgehog.

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Figure 3. Schematic of cardiovascular lineage diversificationThe specification of cardiomyocytes in the first and second heart fields is shown in the

context of other cardiac cells that also derive from a common cardiogenic mesoderm

progenitor. In particular, Isl1 expression distinguishes the first and second heart fields.

Comparison of action potentials for mature myocytes reveals the range of function generated

from each heart field. *Developmental origin of the coronary endothelium is an active topic

of investigation. While some evidence points to partial contributions to the coronary

endothelium from the epicardium70, 160, other sources such as the endocardium161 and the

sinus venouses69 have also been reported.

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Figure 4. Cardiac gene regulatory networkThe diagram shown is a brief overview of a subset of all known transcription factor

interactions and signaling pathways that drive the differentiation of FHF (orange) and SHF

(blue) cardiac progenitor cells during development. Factors colored by both orange and blue

represent regulators of both FHF and SHF. Arrows indicate increased expression of one

transcription factor or signaling molecule due to activity of another transcription factor.

Signaling pathways that activate expression of certain transcription factors are shown in red.

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Figure 5. Specification of chamber myocardiumThe specification of cells to the chamber myocardium is modulated by Tbx5 and Tbx20 in

tandem with more broadly expressed factors Nkx2-5 and Gata4. Tbx2 and Tbx3 suppress

the expression of chamber myocardium-specific genes, resulting in low proliferation rate,

slow conduction velocity, and poor contractibility characteristic to the primary myocardium.

The primary myocardium phenotype becomes restricted to the AVC and p-OFT.

Abbreviations: AVC, atrioventricular canal; p-OFT, proximal outflow tract; d-OFT, distal

outflow tract.

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Figure 6. Spatiotemporal regulation of trabeculationThe development of ventricular trabeculae is governed by signal transduction involving the

endocardium, myocardium, and cardiac jelly. Pathways implicated in trabeculation are

depicted in approximate chronological order of expression from left to right. Beginning with

establishment of the cardiac jelly and endothelium, trabeculation progresses via cellular

proliferation and migration, and ends with degradation of the cardiac jelly. Factors involved

in epigenetic mechanisms are underlined.

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